The extensive replication of nucleic acids, today known as (and referred to herein as) nucleic acid “amplification,” finds wide utility, both practical and theoretical, in a variety of contexts. H. G. Khorana and his co-workers first proposed the use of an in vitro DNA amplification process to increase available amounts of double-stranded DNA (partial sequences of the gene for the major yeast alanine t-RNA) that had been created by the enzymatic ligation of synthetic DNA's. See K. Kleppe et al., J. Mol. Biol. 56:341–361 (1971). Later, in vitro amplification was applied to the amplification of genomic DNA (Saiki et al., Science 230:1350–1354 (1985)) as the technique now known as the polymerase chain reaction or “PCR.” Through the wide availability of synthetic oligonucleotide primers, thermostable DNA polymerases and automated temperature cycling apparatus, PCR became a widely utilized tool of the molecular biologist.
The PCR process is also referred to in the literature as an “exponential amplification” process. Each round or “cycle” of primer extension results in replication of a primer-binding site for the other primer. Thus, each of the synthetic DNA molecules produced in any of the previous cycles is available to serve as a template for primer-dependent replication. This aspect of the process, coupled with the presence of a sufficiently large number of primer molecules, results in synthetic DNA accumulating in a mathematically exponential manner as the reaction proceeds.
PCR has proven to be a valuable technique for the molecular biologist, and has been used extensively in the fields of human genetic research, diagnostics and forensic science, and even in the detection of antibodies. However, disadvantages have nevertheless been recognized. The PCR process can be difficult to quantify accurately, mainly because the amplification products increase exponentially with each round of amplification. The products of PCR, namely, double-stranded DNA molecules, are difficult to analyze or sequence per se. Strand separation typically must be carried out prior to sequencing or other downstream processes that requires single stranded nucleic acids, such as hybridization to a probe capable of detecting the sequence of interest.
The PCR process also has proven to be quite susceptible to contamination generated through the transfer of previously amplified DNA sequences into a new reaction. Such contamination is referred to as carry-over contamination and can cause false-positive results. Carry-over contamination appears to be caused by the facts that (1) very large amounts of DNA are generated in any given reaction cycle, and (2) the process uses all product DNA strands as templates in subsequent cycles. Even minute quantities of contaminating DNA can be exponentially amplified and lead to erroneous results. See Kwok and Higuchi, Nature 339:237–238 (1989). False positive results in a clinical setting can lead to incorrect therapeutic interventions. While useful in any setting, amplification methods that reduce risk of carry-over contamination will have particular utility in clinical diagnostic assays.
As these contamination problems are widely recognized, several approaches have been designed to help limit the risk of product contamination in PCR, including chemical decontamination, utilizing closed systems, use of ultra-violet irradiated work stations (Pao et al, Mol. Cell Probes 7: 217–9 (1993)), cleavable primers (Walder et al., Nucleic Acids Research 21:4, 229–43 (1993)), or enzymatic degradation methods (Longo et al., Gene 93: 125–8 (1990)). None of these methods is totally effective.
A technique that significantly reduces this risk of carry-over contamination has been developed. This technique, linked linear amplification (herein also referred to as “LLA”), uses primers that are modified in such a way that they are, or are rendered, replication defective. Primers that have a blocking group, such as 1,3 propanediol, can support primer extension but cannot be replicated. Therefore, primer extension reactions are terminated when they reach the blocking group, or non-replicable element, of a primer that has been incorporated into a template strand. See, for example, U.S. Pat. Nos. 6,335,184 and 6,027,923. See, also, Reyes et al. Clinical Chemistry 47:1 31–40 (2001); Wu et al. Genomics 4: 560–569 (1989).
Because the primer extension products in LLA cannot serve as a template for subsequent primer binding and primer extension, LLA molecules accumulate in a mathematically linear fashion. The linear accumulation of LLA products renders this process relatively insensitive to carry-over contamination. Although decreased risk of contamination is an advantage of the LLA system, the linear accumulation of LLA products also results in a great disadvantage of this system: LLA requires that an excessive number of reaction cycles and/or primers be used in order to achieve significant amplification. For example, U.S. Pat. No. 6,335,184 discloses that 1,000 cycles would be necessary in order to generate a yield of 500,500 products. Furthermore, Reyes et al., Clinical Chemistry 47:31–40 (2001) discloses that 14 to 18 primers were necessary in order to achieve yields comparable with PCR.
Designing and synthesizing such a large number of primers is time consuming, expensive and difficult. In many cases it may not be possible to obtain the number of functional primers needed for LLA; there simply may not be a sufficient number of acceptable primer-binding sites available.
Obtaining single-stranded product from nucleic acid amplification is particularly useful since many applications for which nucleic acid amplification is employed require a single-stranded nucleic acid. For example, single-stranded nucleic acid amplification products are immediately available for detection by hybridization with a labeled probe. This is particularly useful for diagnostic tests. A single-stranded nucleic acid amplification product can also be immediately sequenced or used as a probe itself. In amplification systems where both strands are equally amplified, such as in traditional PCR and LLA reactions, reannealing of the complementary strands can compete with binding of the labeled probe and interfere with detection of the target, sequencing, probing, etc. Both LLA and PCR require a minimum of 3 primers to yield a single-stranded product and if increased amplification power is desired, more primers may be required to yield a single-stranded product.
Therefore, there is a need for robust nucleic acid amplification systems that produce single-stranded nucleic acid products and pose minimal levels of carry-over contamination.